Inhibition by melittin of phospholipid-sensitive and calmodulin-sensitive Ca2+-dependent protein kinases.

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.     

Stimulation by phspholipid of calcium-dependent phosphorylation of endogenous proteins from mammalian tissues

The occurrence of endogenous substrate proteins for calcium-dependent protein kinase, augmented by either phospholipid or calmodulin, was examined in extracts of several tissues. Pancreas, vas deferens, adrenal and liver were found to contain substrate proteins for phospholipid-sensitive protein kinase. Phosphorylation of pancreatic substrate protein for phospholipid-senstivie protein kinase was rapid and highly sensitive to Ca²⁺, being detectable within 15 s a following exposure to Ca²⁺ and phosphatidylserine and at concentrations of Ca²⁺ as low as 0.5 μM. These findings suggest that the phospholipid-sensitive protien kinase system may serve to mediate some effects of Ca²⁺ in a variety of mammalian cell types.     

Inhibition of mitochondrial oxidative phosphorylation by adriamycin

The antitumour antibiotic, adriamycin, inhibited oxidative phosphorylation in freshly prepared mitochondria from the heart, liver and kidney of the rat. It abolished respiratory control and stimulated ATPase activity. Succinate oxidation by heart mitochondria was extremely sensitive to the drug when hexokinase was present in the reaction medium. The sensitive site has been identified to lie in the region between the succinate dehydrogenase flavoprotein and ubiquinone of the respiratory chain.     

Inhibition by phenothiazine antipsychotic drugs of calcium-dependent phosphorylation of cerebral cortex proteins regulated by phospholipid or calmodulin

Phospholipid-sensitive and calmodulin-sensitive Ca²⁺-dependent phosphorylation of a number of endogenous proteins in the soluble and particulate fractions of the rat cerebral cortex was inhibited by phenothiazine antipsychotic drugs. The mean IC₅₀ values (concentrations causing 50% inhibition of phosphorylation) for trifluoperazine, chlorpromazine and fluphenazine were 16, 24 and 27 μM, respectively. Dibucaine, a local anesthetic drug, was much less effective. It appears that these neuroleptic agents may be useful tools for the study of Ca²⁺-dependent protein phosphorylating systems regulated by either phospholipid or calmodulin in the brain.     

Short-time phosphorylation of plasma membrane proteins by endogenous kinases.

Endogenous protein phosphorylation in plasma membranes isolated from SV 40-transformed mouse fibroblasts was studied in the presence and absence of cyclic nucleotides. Using low concentrations of membrane protein the kinetics of ATP-dependent ³²P-incorporation showed a rapid phosphorylation reaction up to 2 sec of incubation which was stimulated by cAMP and inhibited by cGMP. This short-time phosphorylation reaction was followed by a rapid dephosphorylation and a slower rephosphorylation. This phenomenon was dependent on protein concentration.     

Peptide phosphorylation by calcium-dependent protein kinase from maize seedlings.

Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS15VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cbeta (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L-5X-4R-3X-2X-1SX+1R+2Z+3R+4, where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA.     

Cyclic GMP-dependent phosphorylation of an endogenous protein from rat heart

An endogenous substrate protein (M_r = 70K) for cGMP-dependent protein kinase (G-PK) was found in the cytosol of the rat heart. This protein was specifically phosphorylated by G-PK, with a K_a for cGMP of 0.08 μM, compared with that for cAMP of 2 μM. At least two substrate proteins (M_r = 110K and 26K) specific for cAMP-dependent protein kinase (A-PK) were also noted. The finding in heart of an endogenous substrate protein for G-PK distinguishable from those for A-PK provides additional evidence suggesting an independent role for cGMP in the regulation of cardiac function.     

Inhibition by gossypol of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis

Gossypol, a polyphenolic binaphthalene-dialdehyde extracted from cotton plants which possesses male antifertility action in mammals, is a potent inhibitor of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis. Gossypol inhibited Ca2+-dependent activity of the enzyme without affecting its basal activity. The IC50 value (concentration causing 50% inhibition) was 31 microM when lysine-rich histone was used as substrate. Kinetic analysis indicated that the compound inhibited the enzyme non-competitively with respect to ATP (Ki = 31 microM) or lysine-rich histone (Ki = 30 microM), and competitively with respect to phosphatidylserine (Ki = 2.1 microM). With Ca2+, irrespective of the presence or absence of 1,3-diolein, the compound lowered Vmax and increased the apparent Ka for Ca2+. The compound also inhibited phosphorylation by the enzyme of high-mobility-group 1 protein (one of the endogenous substrates in the testis for the enzyme located in nucleosome), with an IC50 value of 88 microM. These results suggested that a phospholipid-sensitive Ca2+-dependent protein phosphorylation system in the testis is involved in the regulation of spermatogenesis.     

Stimulation of glycogen synthase phosphorylation by calcium-dependent regulator protein.

Phosphorylation of skeletal muscle glycogen synthase catalyzed by a protein kinase is stimulated up to 10-fold by the calcium-dependent regulator (CDR) protein. Half-maximal stimulation requires about 1 microgram of CDR/ml. Phosphorylation by the CDR-dependent synthase kinase is more rapid at pH 8.6 than at pH 6.8 and is blocked by ethylene glycol bis(beta-aminoethyl-ether)N,N'-tetraacetic acid and trifuloperazine. Approximately 60 to 70% of the phosphate is incorporated into the trypsin-insensitive region of glycogen synthase resulting in conversion of the a form to the b form of the enzyme. The CDR-dependent synthase kinase is not myosin light chain kinase, as this enzyme does not phosphorylate glycogen synthase. Furthermore, synthase phosphorylation by the cAMP-dependent protein kinase catalytic subunit is not affected by CDR. The possibility that CDR-dependent synthase kinase may be phosphorylase kinase is being investigated.     

Mitogenic stimulation of thymocytes results in the calcium-dependent phosphorylation of c-ets-1 proteins.

Human, murine and chicken c-ets-1 proteins migrate in SDS-polyacrylamide gels as multiple species. We show here that most if not all of this heterogeneity is due to phosphorylation events occurring predominantly on serine and to a lesser extent on threonine residues. These phosphorylations can be specifically and rapidly stimulated by treatment with the calcium ionophore A23187 or abolished by lowering the extracellular calcium concentration to less than 0.1 microM. The products encoded by c-ets-2 are also phosphorylated in a Ca2+-dependent manner, indicating that these modifications have been conserved in the products encoded by different members of the same gene family. In thymocytes, where the expression of c-ets-1 is elevated as compared with other cell types, c-ets-1 protein phosphorylation occurs after stimulation with mitogenic doses of concanavalin A, is short lived and is strictly dependent upon extracellular Ca2+ sources. This suggests that the c-ets-1 gene product may play a role in the Ca2+-mediated early events linked to T-cell activation.     

Prevention of adriamycin induced heart failure by an increase in endogenous adenosine production

Adenosine (Ado) triggers several protective mechanisms that may attenuate development of heart failure, both locally and systemically. We developed a procedure allowing sustained increase in endogenous Ado production by the combined application of Ado metabolism inhibitors and nucleotide precursors. We found that our procedure attenuate the development of heart failure induced by adriamycin.     

Inhibition of calcium-dependent actin gelation by actin-binding protein from platelets

Various proteins related to cell contraction have been extracted from human platelets. Of these, a protein (48K) with the molecular weight of 48,000 and one with the molecular weight of 47,000 (P47) often migrate together with actin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We studied the biochemical characteristics of the 48K protein, purified by actin affinity and DEAE-Sepharose chromatography. The 48K protein did not react with anti-actin antibody or peroxidase-labelled actin. The protein inhibited the calcium-dependent gelation of actin. The 48K protein seemed to be a regulatory protein involving cell contraction not identified before.     

Subcellular distribution of phospholipid-sensitive calcium-dependent protein kinase in guinea pig heart,spleen and cerebral cortex,and inhibition of the enzyme by Triton X-100

The subcellular distribution of phospholipid-sensitive Ca²⁺-dependent protein kinase in guinea pig heart was found to be: cytosol, 73%; microsome, 18%; plasma membrane, 9%; nuclei and mitochondria, < 0.1%. The enzyme in spleen and cerebral cortex was distributed nearly equally in the cytosolic and total (unfractionated) particulate fractions. The particulate enzyme in heart was released by EGTA (2.5 mM) alone but not by Triton X-100 (0.3%) alone, although a combination of the two was most effective. On the other hand, the particulate enzyme in spleen and cerebral cortex was released only by a combination of Triton X-100 and EGTA. Triton X-100 inhibited the enzyme, and this inhibition was reversed by phosphatidylserine (a phospholipid cofactor for the enzyme). The detergent, however, was without effect on cyclic AMP-dependent and cyclic GMP-dependent protein kinases.     

Polyamines alter the phosphorylation pattern of chromatin proteins by endogenous protein kinases

The effect of polyamines on the chromatin phosphorylation by endogenous protein kinases was investigated. Polyamines not only selectively stimulated the phosphorylation of chromatin proteins but also concurrently inhibited the phosphorylation of a number of polypeptides. In particular, a 11,000-dalton polypeptide with pI 4.5–5.0 was highly phosphorylated in the absence of polyamines, despite being a minor component whereas the phosphorylation was strongly inhibited in the presence of polyamines.     

Inhibition by gossypol of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis

Gossypol, a polyphenolic binaphthalene-dialdehyde extracted from cotton plants which possesses male antifertility action in mammals, is a potent inhibitor of phospholipid-sensitive Ca²⁺-dependent protein kinase from pig testis. Gossypol inhibited Ca²⁺-dependent activity of the enzyme without affecting its basal activity. The IC₅₀ value (concentration causing 50% inhibition) was 31 μM when lysine-rich histone was used as substrate. Kinetic analysis indicated that the compound inhibited the enzyme non-competitively with respect to ATP (K_i = 31 μM) or lysine-rich histone (K_i = 30μM), and competitively with respect to phosphatidylserine (K_i = 2.1 μM). With Ca²⁺, irrespective of the presence or absence of 1,3-diolein, the compound lowered V_max and increased the apparent K_a for Ca²⁺. The compound also inhibited phosphorylation by the enzyme of high-mobility-group 1 protein (one of the endogenous substrate in the testis for the enzyme located in nucleosome), with an IC₅₀ value of 88 μM. These results suggested that a phospholipid-sensitive Ca²⁺-dependent protein phosphorylation system in the testis is involved in the regulation of spermatogenesis.     

Transient state kinetic studies of phosphorylation by ATP and Pi of the calcium-dependent ATPase from sarcoplasmic reticulum.

The ATPase of the sarcoplasmic reticulum is phosphorylated by ATP in the presence of Ca2+. A rapid phosphorylation was observed when the enzyme was preincubated with Ca2+ prior to the addition of 0.1 or 1 mM ATP. The rate of phosphorylation was decreased when Ca2+ was omitted from the preincubation medium and added with ATP when the reaction was started. The rate of phosphorylation by ATP was further decreased when Pi was included in the preincubation medium without Ca2+. In this case, the enzyme was phosphorylated by Pi during the preincubation. When Ca2+ and ATP were added, a burst of phosphorylation by ATP was observed in the initial 16 ms. In the subsequent incubation intervals, the phosphorylation by ATP was synchronous with the hydrolysis of the phosphoenzyme formed by Pi. The rate of hydrolysis of the phosphoenzyme formed by Pi was measured when either the Pi concentration was decreased 10 fold, or when Ca2+, ATP or ATP plus Ca2+ was added to the medium. Upon the single addition of Ca2+, the time for half-maximal decay was in the range 500--1000 ms. In all other conditions it was in the range 70--90 ms.     

Modulation of GT-1 DNA-binding activity by calcium-dependent phosphorylation

The analysis of pea rbcS-3A promoter sequence showed that BoxII was necessary for the control of rbcS-3A gene expression by light. GT-1, a DNA-binding protein that interacts with BoxII in vitro, is a good candidate for being a light-modulated molecular switch controlling gene expression. However, the relationship between GT-1 activity and light-responsive gene activation still remains hypothetical. Because no marked de novo synthesis was detected after light treatment, light may induce post-translational modifications of GT-1 such as phosphorylation or dephosphorylation. Here, we show that recombinant GT-1 (hGT-1) of Arabidopsis can be phosphorylated by various mammalian kinase activities in vitro. Whereas phosphorylation by casein kinase II had no apparent effect on hGT-1 DNA binding, phosphorylation by calcium/calmodulin kinase II (CaMKII) increased the binding activity 10–20-fold. Mass spectrometry analyses of the phosphorylated hGT-1 showed that amongst the 6 potential phosphorylatable residues (T86, T133, S175, T179, S198 and T278), only T133 and S198 are heavily modified. Analyses of mutants altered at T86, T133, S175, T179, S198 and T278 demonstrated that phosphorylation of T133 can account for most of the stimulation of DNA-binding activity by CaMKII, indicating that this residue plays an important role in hGT-1/BoxII interaction. We further showed that nuclear GT-1 DNA-binding activity to BoxII was reduced by treatment with calf intestine phosphatase in extracts prepared from light-grown plants but not from etiolated plants. Taken together, our results suggest that GT-1 may act as a molecular switch modulated by calcium-dependent phosphorylation and dephosphorylation in response to light signals.     

Properties of calcium-dependent regulatory proteins from fungi and yeast

Calmodulins were isolated from vegetative mycelia of Basidiomycetes fungi, Agaricus campestris and Coprinus lagopus. These calmodulins showed similar mobilities to those of animal calmodulins on nondenaturing polyacrylamide gel electrophoresis in the presence or absence of Ca2+. The molecular weights of both calmodulins were determined to be 16,000. Agaricus calmodulin consisted of 148 amino acids including epsilon-N-trimethyllysine and cysteine. The UV-absorption spectrum showed the relatively high content of phenylalanine in Basidiomycetes calmodulins. The difference UV-absorption spectrum due to the blue shift by Ca2+ was observed. Both calmodulins activated muscle myosin light chain kinase and pea NAD+ kinase in a Ca2+-dependent manner, and the activities were inhibited by trifluoperazine or chlorpromazine. A calmodulin-like protein was partially purified from baker's yeast, Saccharomyces cerevisiae. However, detection of a calmodulin-like protein in prokaryotes was not successful.